Abstract The notochord is a defining structure of our phylum Chordata that has critical roles in development that are highly conserved in vertebrates. In addition to acting as an inductive signaling center during early development, the notochord functions as a hydrostatic skeleton. During embryogenesis it provides structural support and participates in anterior-posterior (AP) axis elongation. Later in development, the notochord serves as a scaffold for vertebral column formation. The notochord's mechanical properties depend on its unique structure. The notochord is composed of an inner core of large (~40 m) fluid filled vacuolated cells surrounded by epithelial- like sheath cells. The entire structure is encased by an elaborate and semi-rigid extracellular matrix. Previous research from our lab has shown that during early development, the vacuolated cells rapidly expand within the inelastic notochord sheath through the inflation of their intracellular vacuole. The incompressible nature of the fluid within the vacuoles and the rigidity of the external sheath lead to expansion of the rod along the axis, driving embryonic AP elongation. Loss of vacuolated cells or vacuole fragmentation results in a shorter embryonic axis and a kinked vertebral column. However, the precise cellular and molecular processes by which large vacuolated cells of the notochord maintain integrity while being subjected to significant stresses remain unknown. Previous reports have shown that vacuolated cells posses a high density of invaginations at the cell surface known as caveolae and that these structures may play a role in notochord development. On the other hand, preliminary data from our laboratory suggest that the mechano-sensitive calcium channel Trpv4 may function during vacuolated cell expansion. I will determine if developing vacuolated cells utilize both of these plasma membrane components to buffer rapid changes in cell volume and control vacuole dynamics. These studies will elucidate novel mechanisms of cell surface integrity and maintenance, which are broadly applicable to many cell types. Importantly, my work with physiologically relevant zebrafish models will provide insight on how proper vacuolated cell inflation leads to a structurally intact notochord. The proposed studies are relevant to human health and will inform our understanding of vertebral column development.